US9005986B2 - Method for rapidly measuring melatonin adulteration of Chinese patent medicine or healthcare food - Google Patents

Method for rapidly measuring melatonin adulteration of Chinese patent medicine or healthcare food Download PDF

Info

Publication number
US9005986B2
US9005986B2 US14/346,532 US201214346532A US9005986B2 US 9005986 B2 US9005986 B2 US 9005986B2 US 201214346532 A US201214346532 A US 201214346532A US 9005986 B2 US9005986 B2 US 9005986B2
Authority
US
United States
Prior art keywords
melatonin
chinese patent
dimethylaminocinnamaldehyde
patent medicine
hydrochloric acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
US14/346,532
Other versions
US20140220698A1 (en
Inventor
Tiejie Wang
Yi Lu
Lihe Xiao
Xiaoying Guan
Dongqi Han
Guo Yin
Jue Wang
Xueqing Li
Yan Yan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute for Drug Control
Original Assignee
Shenzhen Institute for Drug Control
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Institute for Drug Control filed Critical Shenzhen Institute for Drug Control
Assigned to Shenzhen Institute for Drug Control reassignment Shenzhen Institute for Drug Control ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GUAN, XIAOYING, HAN, Dongqi, LI, XUEQING, LU, YI, WANG, JUE, WANG, TIEJIE, XIAO, LIHE, YAN, YAN, YIN, Guo
Publication of US20140220698A1 publication Critical patent/US20140220698A1/en
Application granted granted Critical
Publication of US9005986B2 publication Critical patent/US9005986B2/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/145555—Hetero-N
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

  • the present invention relates to a method for measuring melatonin components, specifically for detecting whether there is melatonin added to Chinese patent medicine or healthcare food, especially to sedative hypnotic Chinese patent medicine or healthcare food.
  • Melatonin also known chemically as N-acetyl-5-methoxytryptamine, is a kind of indoleamine hormone secreted by the pineal gland located in the human brain. Known as the “hormone of darkness”, melatonin secretion decreases with increasing intensity of light. Melatonin is mainly used to regulate sleep-wake cycle and physiological circadian rhythms. Melatonin also has anti-aging properties.
  • Melatonin is a kind of endogenous hormone. Some studies have shown that heavy use of melatonin may cause breathing problems and hangover effect. Meanwhile, melatonin may impact on some sedative hypnotic drug's efficacy, such as clonazepam. In Europe, dosage interval, target population and dosage are all strictly restricted. In the US, melatonin can be sold in the form of dietary supplements. The label must include content and dosage instructions. In China, melatonin is used as healthcare food.
  • melatonin daily dosage restriction of melatonin is 1-3 mg; no ingredients can be added except vitamin B6; the section of “warning” should tell the people who engage in mechanical work or dangerous operation not to use before they work/while they are working; meanwhile, it should also tell the people having autoimmune disease (rheumatism, etc.) or hyperthyroidism disease to use with caution. Thus, melatonin should be consumed according to doctor's advice. The dosage must be confirmed. Melatonin should be used with more caution in children. However, due to good sedative hypnotic effect of melatonin and complex ingredients of Chinese patent medicine and healthcare food, some unscrupulous people add melatonin to sedative hypnotic Chinese patent medicine or healthcare food illegally.
  • the existing methods for detecting whether there is melatonin added to sedative hypnotic Chinese patent medicine or healthcare food mainly include the following four methods:
  • the component to be tested is extracted from samples with methanol.
  • a small spot of test solution is applied to a silica gel plate, as well as reference solution.
  • Developing agent is used to develop the plate.
  • the plate is taken out, dried, and inspected under ultraviolet lamp at 254 nm.
  • different substances appear at different TLC plate position in the form of spot due to the differences in migration rates.
  • the presence of target components in the samples can be determined by the experimental results that the spot of test sample appears at the same position as the reference. If spot appears on TLC chromatogram of test solution is at the same position as melatonin reference solution, it may indicate the presence of melatonin in the test sample.
  • TLC is free of expensive analytical instrument. Disadvantages are: (1) Low in resolution; traditional Chinese medicine are complex, there are a lot of interference. Some coexisting components can be easily mistaken for melatonin If some coexisting components are in a large amount and chromatographic shift value is close to that of the component to be tested, the detection of target components may be interfered. (2) It takes long time to develop and dry, which fails to meet the requirement of rapid measurement. (3) TLC plate needs to be dried and stored in dry place. Environment temperature and humidity have a great effect on separation. (4) Require fixed location. Not suitable for mobile on-site detection. (5) Require tester with rich experience
  • HPLC is commonly used modern analytical method. Under the same chromatographic conditions, different materials have different retention time. Under the same chromatographic conditions, melatonin reference solution and test solution extracted from the sample are injected separately. According to the retention time of chromatographic peak, it can be determined whether the test sample contains melatonin components.
  • HPLC-MS using HPLC as separator, mass spectrography as analyzer, is suitable to analyze complex composition, even in the case of serious interference. This analytical method can improve the reliability of the test results.
  • HPLC-MS is an analytical method, costing more and being more complex than HPLC. Instrument has higher requirements to the environment. Instrument needs to be fixed, and is not suitable for mobile on-site detection. These detections can only be performed in a few laboratories, so it is difficult to promote.
  • the object of the present invention is to provide a low-cost method for rapidly measuring melatonin.
  • the method overcomes deficiencies of the current measurement technique. There is no need to use expensive analytical instrument.
  • the method has strong specificity, high accuracy, reaction sensitivity, and is applicable to on-site detection of melatonin adulteration of Chinese patent medicine or healthcare food.
  • Another object of the present invention is to provide use of the above method in preparing melatonin test kit.
  • Yet another object of the present invention is to provide use of the above method in screening melatonin adulteration of Chinese patent medicine or healthcare food.
  • the method for rapidly measuring melatonin adulteration of Chinese patent medicine or healthcare food comprising the following steps in sequence:
  • Melatonin also known chemically as N-acetyl-5-methoxytryptamine, is a kind of indoleamine hormone. Our studies show that melatonin solution becomes blue-green in color after adding hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde.
  • Solid Chinese patent medicine or healthcare food is grinded, followed by addition of 1-3 mL of ethyl acetate. After shaking for 1 min, the mixture obtained from the previous step is filtrated to get test liquid; or 1-3 mL of ethyl acetate is added to liquid Chinese patent medicine or healthcare food. After shaking for 1 min and standing, then filtrate the ethyl acetate phase to get test liquid.
  • oral capsule tablet pill granules liquid Amount of 1 capsule 1 tablet 1 pill 1/10 1 ⁇ 5 vial samples package
  • hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde 10 mL of hydrochloric acid ethanol solution having a mass concentration of 40-70% is added to 0.01-0.1 g of p-dimethylaminocinnamaldehyde in order to dissolve p-dimethylaminocinnamaldehyde.
  • Concentration of the hydrochloric acid ethanol solution herein is preferably in the range of 50%-60%.
  • the amount of p-dimethylaminocinnamaldehyde and volume of hydrochloric acid ethanol solution can be adjusted according to the actual situation, but p-dimethylaminocinnamaldehyde and hydrochloric acid ethanol solution are in an amount sufficient to get a hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde having a concentration of 1-10 mg/mL.
  • Ratio of hydrochloric acid to ethanol herein is preferably to be in the range of 1.5:1 to 1:1. More preferably, color developing agent is prepared using hydrochloric acid-ethanol in a ratio of 1:1. The color rendering is excellent when concentration of p-dimethylaminocinnamaldehyde is in the range of 4-6 mg/mL. More preferably, the concentration of hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde is 4 mg/mL.
  • melatonin test kit can also be prepared.
  • the method can be applied in screening melatonin adulteration of Chinese patent medicine or healthcare food.
  • the present invention has the following advantages:
  • the method can be applied for liquid sample and solid sample.
  • the method can be applied for both traditional Chinese medicines and healthcare food.
  • the material used in the present invention is low in cost, and suitable for popularization and large-scale production.
  • the method for measuring melatonin of the present invention is also suitable for rapidly measuring melatonin adulteration of other foods having effects on insomnia and sleep.
  • a grain of the containers of such healthcare food A was placed into sample cell. 2 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
  • a grain of the containers of such healthcare food B was placed into sample cell. 3 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 60% used as color developing agent was added to 0.08 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 0.5 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
  • a grain of the containers of such healthcare food C was placed into sample cell. 3 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 40% used as color developing agent was added to 0.1 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 0.5 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
  • a grain of the containers of such healthcare food D was placed into sample cell. 3 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 40% used as color developing agent was added to 0.01 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 0.5 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
  • a grain of the containers of such healthcare food E was placed into sample cell. 1 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 70% used as color developing agent was added to 0.05 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 0.6 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
  • a tablet of such healthcare food F was grinded and placed into sample cell. 2 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The color of the solution did not change within 30 s. This indicated that the sample contains no melatonin.
  • a grain of the containers of such Chinese patent medicine G was placed into sample cell. 3 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The color of the solution did not change within 30 s. This indicated that the sample contains no melatonin
  • a pill of such Chinese patent medicine H was grinded and placed into sample cell. 1 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The color of the solution did not change within 30 s. This indicated that the sample contains no melatonin.
  • Tests were carried out on 80 samples, which consist of Chinese patent medicine having sedative and tranquilization effect, healthcare food having the function of improving sleep and healthy products having the same function according to the label instructions. The test results were verified by related verification method and shown in Table. 1:
  • the method can be used to for rapidly screening melatonin adulteration of healthy products.
  • the method is rapid, simple and convenient, has strong specificity, high accuracy, reaction sensitivity, and a wide application range, and is applicable to on-site detection of melatonin as required by grass root inspection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Endocrinology (AREA)
  • Plasma & Fusion (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Medicinal Preparation (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)

Abstract

A method for rapidly measuring melatonin adulteration of Chinese patent medicines and healthcare foods comprises: (1) extracting melatonin added to a Chinese patent medicine or healthcare food by using ethyl acetate; and (2) adding p-dimethylaminocinnamaldehyde to the extracted solution, and observing color. The method is rapid, simple and convenient, has strong specificity, high accuracy, reaction sensitivity, and a wide application range, and is applicable to on-site detection of melatonin adulteration of a Chinese patent medicine or healthcare food.

Description

PRIORITY INFORMATION
The present application claims priority to Chinese Application No. 201110316788.3, entitled Method for Rapidly Measuring Melatonin Adulteration of Chinese Patent Medicine or Healthcare Food, filed on Oct. 18, 2011, the entire contents of which are hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to a method for measuring melatonin components, specifically for detecting whether there is melatonin added to Chinese patent medicine or healthcare food, especially to sedative hypnotic Chinese patent medicine or healthcare food.
BACKGROUND OF THE INVENTION
Currently, there are many commercially available Chinese patent medicines or healthcare foods that promise to solve the problem of insomnia, which is also declared to be composed of pure plant or pure traditional Chinese medicine, do not contain Western medicine used to treat sleep problems or hormones.
Melatonin, also known chemically as N-acetyl-5-methoxytryptamine, is a kind of indoleamine hormone secreted by the pineal gland located in the human brain. Known as the “hormone of darkness”, melatonin secretion decreases with increasing intensity of light. Melatonin is mainly used to regulate sleep-wake cycle and physiological circadian rhythms. Melatonin also has anti-aging properties.
Melatonin is a kind of endogenous hormone. Some studies have shown that heavy use of melatonin may cause breathing problems and hangover effect. Meanwhile, melatonin may impact on some sedative hypnotic drug's efficacy, such as clonazepam. In Europe, dosage interval, target population and dosage are all strictly restricted. In the US, melatonin can be sold in the form of dietary supplements. The label must include content and dosage instructions. In China, melatonin is used as healthcare food. There are clear legal provisions: daily dosage restriction of melatonin is 1-3 mg; no ingredients can be added except vitamin B6; the section of “warning” should tell the people who engage in mechanical work or dangerous operation not to use before they work/while they are working; meanwhile, it should also tell the people having autoimmune disease (rheumatism, etc.) or hyperthyroidism disease to use with caution. Thus, melatonin should be consumed according to doctor's advice. The dosage must be confirmed. Melatonin should be used with more caution in children. However, due to good sedative hypnotic effect of melatonin and complex ingredients of Chinese patent medicine and healthcare food, some unscrupulous people add melatonin to sedative hypnotic Chinese patent medicine or healthcare food illegally. They hype the effect of the products in order to drive up prices. It makes users not clearly aware of their dosage, thus causing adverse consequences and harm to the people's health. Especially in some rural area and underdeveloped small cities, lack of necessary testing equipment and weak technical regulation has created opportunities for criminals to employ trickery, resulting in a large number of counterfeit or substandard drugs, causing confusion in the market of traditional Chinese medicine and healthcare food.
The existing methods for detecting whether there is melatonin added to sedative hypnotic Chinese patent medicine or healthcare food mainly include the following four methods:
1. Thin Layer Chromatography (TLC)
The component to be tested is extracted from samples with methanol. A small spot of test solution is applied to a silica gel plate, as well as reference solution. Developing agent is used to develop the plate. Then the plate is taken out, dried, and inspected under ultraviolet lamp at 254 nm. With the help of developing agent, different substances appear at different TLC plate position in the form of spot due to the differences in migration rates. The presence of target components in the samples can be determined by the experimental results that the spot of test sample appears at the same position as the reference. If spot appears on TLC chromatogram of test solution is at the same position as melatonin reference solution, it may indicate the presence of melatonin in the test sample.
Advantage of TLC is free of expensive analytical instrument. Disadvantages are: (1) Low in resolution; traditional Chinese medicine are complex, there are a lot of interference. Some coexisting components can be easily mistaken for melatonin If some coexisting components are in a large amount and chromatographic shift value is close to that of the component to be tested, the detection of target components may be interfered. (2) It takes long time to develop and dry, which fails to meet the requirement of rapid measurement. (3) TLC plate needs to be dried and stored in dry place. Environment temperature and humidity have a great effect on separation. (4) Require fixed location. Not suitable for mobile on-site detection. (5) Require tester with rich experience
2. High Performance Liquid Chromatography (HPLC)
HPLC is commonly used modern analytical method. Under the same chromatographic conditions, different materials have different retention time. Under the same chromatographic conditions, melatonin reference solution and test solution extracted from the sample are injected separately. According to the retention time of chromatographic peak, it can be determined whether the test sample contains melatonin components.
Advantages of HPLC chromatography is high efficiency and high sensitivity. Disadvantages are: (1) The equipment is expensive; it takes long time for pretreatment of sample; the column is vulnerable to be polluted; high cost; (2) It is easy to make a mistake in judgment when the peak retention time of some coexisting components is close to that of melatonin; (3) Instrument has high requirements to the environment, a fixed location is required; not suitable for mobile on-site detection.
3. High Performance Liquid Chromatography—Mass Spectrography (HPLC-MS)
HPLC-MS, using HPLC as separator, mass spectrography as analyzer, is suitable to analyze complex composition, even in the case of serious interference. This analytical method can improve the reliability of the test results.
HPLC-MS is an analytical method, costing more and being more complex than HPLC. Instrument has higher requirements to the environment. Instrument needs to be fixed, and is not suitable for mobile on-site detection. These detections can only be performed in a few laboratories, so it is difficult to promote.
In the field of Chinese patent medicine and healthcare food, the method for rapidly screening melatonin by physical and chemical reaction has not been reported yet. For drugstore and grassroots drug test department, it is very necessary to develop a method for rapidly screening melatonin adulteration of Chinese patent medicine or healthcare food. It will help on-site detection and provide evidence timely for regulation of Chinese patent medicine or healthcare food. It is useful to combat counterfeiting and protect the medication safety of the people.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a low-cost method for rapidly measuring melatonin. The method overcomes deficiencies of the current measurement technique. There is no need to use expensive analytical instrument. The method has strong specificity, high accuracy, reaction sensitivity, and is applicable to on-site detection of melatonin adulteration of Chinese patent medicine or healthcare food.
Another object of the present invention is to provide use of the above method in preparing melatonin test kit.
Yet another object of the present invention is to provide use of the above method in screening melatonin adulteration of Chinese patent medicine or healthcare food.
The method for rapidly measuring melatonin adulteration of Chinese patent medicine or healthcare food, comprising the following steps in sequence:
(1) extracting melatonin from Chinese patent medicine or healthcare food with ethyl acetate;
(2) adding p-dimethylaminocinnamaldehyde to extracted solution as color developing agent.
Melatonin, also known chemically as N-acetyl-5-methoxytryptamine, is a kind of indoleamine hormone. Our studies show that melatonin solution becomes blue-green in color after adding hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde.
Figure US09005986-20150414-C00001
The technical solution is as follows:
1. Solid Chinese patent medicine or healthcare food is grinded, followed by addition of 1-3 mL of ethyl acetate. After shaking for 1 min, the mixture obtained from the previous step is filtrated to get test liquid; or 1-3 mL of ethyl acetate is added to liquid Chinese patent medicine or healthcare food. After shaking for 1 min and standing, then filtrate the ethyl acetate phase to get test liquid.
In relation to most commercially available Chinese patent medicine and healthcare food, recommended amount of samples are as follows:
Dosage form
oral
capsule tablet pill granules liquid
Amount of 1 capsule 1 tablet 1 pill 1/10 ⅕ vial
samples package
2. Preparation of hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde: 10 mL of hydrochloric acid ethanol solution having a mass concentration of 40-70% is added to 0.01-0.1 g of p-dimethylaminocinnamaldehyde in order to dissolve p-dimethylaminocinnamaldehyde. Concentration of the hydrochloric acid ethanol solution herein is preferably in the range of 50%-60%. The amount of p-dimethylaminocinnamaldehyde and volume of hydrochloric acid ethanol solution can be adjusted according to the actual situation, but p-dimethylaminocinnamaldehyde and hydrochloric acid ethanol solution are in an amount sufficient to get a hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde having a concentration of 1-10 mg/mL.
Ratio of hydrochloric acid to ethanol herein is preferably to be in the range of 1.5:1 to 1:1. More preferably, color developing agent is prepared using hydrochloric acid-ethanol in a ratio of 1:1. The color rendering is excellent when concentration of p-dimethylaminocinnamaldehyde is in the range of 4-6 mg/mL. More preferably, the concentration of hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde is 4 mg/mL.
3. 0.5-1 mL of hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde is added to the test liquid. If the solution turns blue-green rapidly (exhibiting obvious blue to green in color), the sample contains melatonin; if no obvious blue-green can be observed after 30 s (no obvious blue to green in color), the sample contains no melatonin
According to the present invention, melatonin test kit can also be prepared.
According to the present invention, the method can be applied in screening melatonin adulteration of Chinese patent medicine or healthcare food.
Compared to the prior art, the present invention has the following advantages:
(1) Rapid and simple: It takes 1 minute for extraction, 30 seconds for color reaction. The whole process can be completed within 2 minutes. The method is much simpler than liquid chromatography and LC-MS (require at least 2-3 hours). Besides, there is no need to use reference substance.
(2) Strong in specificity and high in accuracy: according to the verified test results, commonly used excipients in tablets, capsules, pills, granules, oral liquid do not interfere with the detection of melatonin.
(3) High in reaction sensitivity: The minimum detection limit of such method is 1/200 of efficient dosage in clinic (average detection limit for the samples is 0.025 mg/ml). Therefore, the method can be used for sensitive detection of melatonin in Chinese patent medicine or healthcare food.
(4) Covers a wide range of application: The method can be applied for liquid sample and solid sample. The method can be applied for both traditional Chinese medicines and healthcare food.
(5) Economical: the material used in the present invention is low in cost, and suitable for popularization and large-scale production.
The method for measuring melatonin of the present invention is also suitable for rapidly measuring melatonin adulteration of other foods having effects on insomnia and sleep.
DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS
While the present invention has been described with reference to particular embodiments, it will be understood that the embodiments are illustrative and that the invention scope is not so limited.
EXAMPLE 1
Healthcare food A purchased from the market (Capsule, dosage according to the label instructions was 1-2 capsules every time. The samples were tested by HPLC, each capsule contains melatonin 1 mg.)
A grain of the containers of such healthcare food A was placed into sample cell. 2 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
EXAMPLE 2
Healthcare food B purchased from the market (Capsule, dosage according to the label instructions was 1-2 capsules every time. The samples were tested by HPLC, each capsule contains melatonin 0.9 mg.)
A grain of the containers of such healthcare food B was placed into sample cell. 3 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 60% used as color developing agent was added to 0.08 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 0.5 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
Example 3
Healthcare food C purchased from the market (Capsule, dosage according to the label instructions was 1-6 capsules every time. The samples were tested by HPLC, each capsule contains melatonin 1 mg.)
A grain of the containers of such healthcare food C was placed into sample cell. 3 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 40% used as color developing agent was added to 0.1 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 0.5 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
EXAMPLE 4
Healthcare food D purchased from the market (Capsule, dosage according to the label instructions was 2 capsules every time. The samples were tested by HPLC, each capsule contains melatonin 1.8 mg.)
A grain of the containers of such healthcare food D was placed into sample cell. 3 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 40% used as color developing agent was added to 0.01 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 0.5 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
EXAMPLE 5
Healthcare food E purchased from the market (Capsule, dosage according to the label instructions was 2 capsules every time. The samples were tested by HPLC, each capsule contains melatonin 0.9 mg.)
A grain of the containers of such healthcare food E was placed into sample cell. 1 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 70% used as color developing agent was added to 0.05 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 0.6 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The solution turned blue-green within 30 s. This indicated that the sample contains melatonin illegally.
EXAMPLE 6
Healthcare food F purchased from the market (Tablet, dosage according to the label instructions was 2 tablets every time. The samples were tested to be melatonin-free by HPLC-MS, for negative control test use.)
A tablet of such healthcare food F was grinded and placed into sample cell. 2 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The color of the solution did not change within 30 s. This indicated that the sample contains no melatonin.
EXAMPLE 7
Chinese patent medicine G purchased from the market (Capsule, dosage according to the label instructions was 4 capsules every time. The samples were tested to be melatonin-free by HPLC-MS, for negative control test use.)
A grain of the containers of such Chinese patent medicine G was placed into sample cell. 3 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The color of the solution did not change within 30 s. This indicated that the sample contains no melatonin
EXAMPLE 8
Chinese patent medicine H purchased from the market (Pill, dosage according to the label instructions was about 28 pills every time. The samples were tested to be melatonin-free by HPLC-MS, for negative control test use.)
A pill of such Chinese patent medicine H was grinded and placed into sample cell. 1 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The color of the solution did not change within 30 s. This indicated that the sample contains no melatonin.
EXAMPLE 9
Chinese patent medicine I purchased from the market (Oral liquid, dosage according to the label instructions was 10-20 mL every time. The samples were tested to be melatonin-free by HPLC-MS, for negative control test use.)
2 mL of such Chinese patent medicine I was placed into sample cell. 2 mL of Ethyl acetate was added. After shaking for 1 min and standing, ethyl acetate phase was taken and filtrated, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The color of the solution did not change within 30 s. This indicated that the sample contains no melatonin.
EXAMPLE 10
Chinese patent medicine J purchased from the market (Granules, dosage according to the label instructions was 5 g every time. The samples were tested to be melatonin-free by HPLC-MS, for negative control test use.)
0.5 g of such Chinese patent medicine J was grinded and placed into sample cell. 3 mL of Ethyl acetate was added. After shaking for 1 min and filtrating, the filtrate liquid was used as test liquid. 10 mL of hydrochloric acid ethanol solution having a mass concentration of 50% used as color developing agent was added to 0.04 g of p-dimethylaminocinnamaldehyde to dissolve p-dimethylaminocinnamaldehyde. 1 mL of Hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde was added to the test liquid. The color of the solution did not change within 30 s. This indicated that the sample contains no melatonin.
Rapid Screening Method Assessment
Tests were carried out on 80 samples, which consist of Chinese patent medicine having sedative and tranquilization effect, healthcare food having the function of improving sleep and healthy products having the same function according to the label instructions. The test results were verified by related verification method and shown in Table. 1:
TABLE 1
Verified test results
Rapid
screening Final result
result Melatonin-added Melatonin-free Total
positive True positive A (25) False positive B (1) 26
negative False negative C (0) True negative D (54) 54
Total 25 55 80
As requested by Guangdong Institute for Drug Control, according to the “Guidelines and Technical Requirements to Method for Rapidly Screening Chemical Components Adulteration of Chinese Patent Medicine or Healthcare Food (for trial implementation)”, summary table of the rapid screening method is shown in Table. 2:
TABLE 2
summary table of the Rapid screening method
Sample source 80 samples collected from market (purchased
from the market, sampling from circulation)
there are 25 positive samples
Dosage form Capsule, tablet, granules, pill, oral liquid
Minimum detection The minimum detection limit of such method is
limit one twentieth of efficient dosage in clinic.
Sensitivity  100%
Specificity 98.1%
Missing rate   0%
False rate  1.9%
Accuracy 98.7%
Youden's index 98.7%
Testing time Less than 2 minutes per sample
Professional skill Simply trained
requirements
Operating Operate on site
conditions
It can be seen that the method can be used to for rapidly screening melatonin adulteration of healthy products. The method is rapid, simple and convenient, has strong specificity, high accuracy, reaction sensitivity, and a wide application range, and is applicable to on-site detection of melatonin as required by grass root inspection.

Claims (5)

What is claimed is:
1. A method for rapidly measuring melatonin adulteration of Chinese patent medicine or healthcare food, comprising the following steps in sequence:
(1) obtaining a sample of Chinese patent medicine/healthcare food suspected of having been adulterated with melatonin;
(2) adding ethyl acetate to the sample to form a test liquid;
(3) adding p-dimethylaminocinnamaldehyde and a hydrochloric acid ethanol solution to the test liquid;
(4) and observing if there is a change in color in the test liquid that the Chinese patent medicine or healthcare food has been adulterated with melatonin.
2. The method according to claim 1, wherein mass concentration of said hydrochloric acid ethanol solution is in the range of 40%-70%.
3. The method according to claim 2, wherein concentration of said p-dimethylaminocinnamaldehyde in the hydrochloric acid ethanol solution is in the range of 1-10 mg/mL.
4. The method according to claim 1, comprising the following steps in sequence:
(1) grinding Chinese patent medicine or healthcare food, adding ethyl acetate and shaking, filtrating, taking filtrate liquid as the test liquid;
(2) taking 0.01-0.1 g of p-dimethylaminocinnamaldehyde, adding 10 mL of a hydrochloric acid ethanol solution having a mass concentration of 40%-70% used as color developing agent to dissolve p-dimethylaminocinnamaldehyde;
(3) adding a hydrochloric acid ethanol solution of p-dimethylaminocinnamaldehyde to said test liquid, and observing if there is a change in color that the Chinese patent medicine or healthcare food has been adulterated with melatonin.
5. The method according to claim 1, wherein concentration of said p-dimethylaminocinnamaldehyde in the hydrochloric acid ethanol solution is in the range of 1-10 mg/mL.
US14/346,532 2011-10-18 2012-07-02 Method for rapidly measuring melatonin adulteration of Chinese patent medicine or healthcare food Active US9005986B2 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN2011103167883A CN102393391B (en) 2011-10-18 2011-10-18 Rapid determination method of melatonin adulterated in Chinese patent medicine and health food
CN201110316788.3 2011-10-18
CN201110316788 2011-10-18
PCT/CN2012/078069 WO2013056572A1 (en) 2011-10-18 2012-07-02 Method for rapidly measuring melatonin adulteration of chinese patent medicine or healthcare food

Publications (2)

Publication Number Publication Date
US20140220698A1 US20140220698A1 (en) 2014-08-07
US9005986B2 true US9005986B2 (en) 2015-04-14

Family

ID=45860751

Family Applications (1)

Application Number Title Priority Date Filing Date
US14/346,532 Active US9005986B2 (en) 2011-10-18 2012-07-02 Method for rapidly measuring melatonin adulteration of Chinese patent medicine or healthcare food

Country Status (3)

Country Link
US (1) US9005986B2 (en)
CN (1) CN102393391B (en)
WO (1) WO2013056572A1 (en)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102393391B (en) * 2011-10-18 2013-02-27 深圳市药品检验所 Rapid determination method of melatonin adulterated in Chinese patent medicine and health food
CN103278499A (en) * 2013-05-28 2013-09-04 无锡中德伯尔生物技术有限公司 Method for testing melatonin
CN104237230B (en) * 2014-10-14 2017-12-26 厦门奥亚仪器有限公司 The quick determination method of epiphysin and its operation vessel combination
CN108982490A (en) * 2018-07-10 2018-12-11 江苏苏博检测技术有限公司 A kind of rapid detection method and detection kit of epiphysin
CN109239063A (en) * 2018-09-29 2019-01-18 杭州天迈生物科技有限公司 A kind of rapid detection method of melatonin in health products
CN116076716B (en) * 2023-01-18 2024-12-03 中南林业科技大学 Melatonin-enriched food extract and method of producing the same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6806090B1 (en) * 1999-02-10 2004-10-19 Oxford Natural Products Plc Process for quality control and standardization of medicinal plant products

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5146927A (en) * 1991-06-14 1992-09-15 Brigham And Women's Hospital Test for evaluation of visual functioning in visually impaired subjects
US6562858B2 (en) * 1999-05-17 2003-05-13 St. Elizabeth's Medical Center Of Boston, Inc. Method for treating depression
JP2001165858A (en) * 1999-12-06 2001-06-22 Bunshi Biophotonics Kenkyusho:Kk Method and apparatus for measuring 5-alkoxyindoles
CN1151128C (en) * 2002-05-15 2004-05-26 张绍文 Process for preparing pineal hormone
WO2005078444A1 (en) * 2004-02-18 2005-08-25 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with human melatonin receptor type 1b (mtnr1b)
CN100333651C (en) * 2004-09-03 2007-08-29 光明乳业股份有限公司 Method for purifying melatomin in milk product and detection method therefor
CN102393391B (en) * 2011-10-18 2013-02-27 深圳市药品检验所 Rapid determination method of melatonin adulterated in Chinese patent medicine and health food

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6806090B1 (en) * 1999-02-10 2004-10-19 Oxford Natural Products Plc Process for quality control and standardization of medicinal plant products

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
http://www.google.com/patents/CN1742588A?cl=en. *

Also Published As

Publication number Publication date
CN102393391B (en) 2013-02-27
WO2013056572A1 (en) 2013-04-25
US20140220698A1 (en) 2014-08-07
CN102393391A (en) 2012-03-28

Similar Documents

Publication Publication Date Title
US9005986B2 (en) Method for rapidly measuring melatonin adulteration of Chinese patent medicine or healthcare food
CN102854281B (en) Detection method of sugar-free strong loquat syrup
CN102269751B (en) Detection method of Liuweinengxiao preparation
CN101581712A (en) Quick test method of biguanide compounds added in hypoglycemic medicine, health care products or food and application thereof
CN103969454B (en) Melatonin method for quick and detection thereof block and preparation method
CN105510312B (en) A kind of method for quick of orlistat
Wang et al. Determination of sildenafil by preconcentration on surfactant coated polymeric resin followed by spectrofluorimetry
CN102759598B (en) Quality detection method for 29-componnet stagnation dissipation powder as Tibetan medicinal composition and preparations thereof
CN101408519B (en) A method for detecting sibutramine hydrochloride and its application
CN104777244B (en) A kind of quality determining method of Xingqi Tannikaer capsule
CN103645179A (en) Method for rapidly detecting sulfonylomocznik compound added in hypoglycemic health foods, Chinese patent medicines and foodstuff and applications thereof
CN104237230B (en) The quick determination method of epiphysin and its operation vessel combination
CN102565046B (en) Method for rapidly detecting mifepristone
US20230067029A1 (en) Arylalkylamine, pyrrole, indole, and opiate derivative concentration determination method and test kit using said method
CN109239063A (en) A kind of rapid detection method of melatonin in health products
CN103278498A (en) Detecting method and kit of sibutramine hydrochloride
CN101365952B (en) Assay, kit and apparatus for detection of artemisinin derivatives
CN104297242B (en) Quickly detection vitamin E or the method containing Vitamin E preparation
CN104483445B (en) A kind of thiazolidinedione quick detection kit and detection method thereof
CN102426213B (en) A detection method for Xueshan Weibao Pills
CN101692051B (en) Kit for detecting chemical antihypertensive compositions doped in Chinese herbal medicinal antihypertensive product
CN100387986C (en) A kind of quality control method of Zhenju Jiangya tablet
CN106525842A (en) Quick detection method of dexamethasone
CN102023157A (en) Quick measurement method for pyrazolone substances doped in medicaments and health-care food
Riccio et al. ANALYSIS OF SYNTHETIC ADULTERANTS, MEAN WEIGHT AND LABELING IN TRADITIONAL PHYTOTHERAPIC PRODUCTS

Legal Events

Date Code Title Description
AS Assignment

Owner name: SHENZHEN INSTITUTE FOR DRUG CONTROL, CHINA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WANG, TIEJIE;LU, YI;XIAO, LIHE;AND OTHERS;REEL/FRAME:032498/0614

Effective date: 20140305

STCF Information on status: patent grant

Free format text: PATENTED CASE

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YR, SMALL ENTITY (ORIGINAL EVENT CODE: M2551); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

Year of fee payment: 4

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 8TH YR, SMALL ENTITY (ORIGINAL EVENT CODE: M2552); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

Year of fee payment: 8